A number of plasmid-encoded mechanisms involved in host-mediated insensitivity to phage attack in the lactic streptococci have been described in the literature. The insensitivity may be at the level of adsorption, restriction-modification (R-M) of phage DNA, or by powerful, but as yet uncharacterized, mechanisms that prevent phage maturation.
Two plasmid-encoded adsorption blocking systems have been described by Sanders and Klaenhammer (19) and de Vos et al (20). These molecules, pME0030 (30 Mdal. from S. lactis ME2, ref. 19) and pSKI12 (34 Mdal, from S. cremoris SKII, ref. 20) have the effect of significantly inhibiting phage adsorption to plasmid-containing hosts with a consequent suppression of phage replication. Elimination of the plasmids from their respective hosts results in highly efficient phage adsorption and an increased sensitivity to specific phage. However, since neither plasmid was reported to be conjugative, it may be difficult to exploit them to construct new phage insensitive starter culture strains.
Plasmids encoding R-M activity, some of which are conjugative, occur widely in lactic streptococci (21, 22, 23, 24, 25, 26, 27, 28). Although powerful R-M systems have been achieved by the additive effect of two plasmids (21, 24) the potential of R-M as a defence mechanism against phage is limited since the insensitivity conferred is not complete and phage which become modified (i.e. protected from subsequent restriction) pose a threat to the host that previously restricted the unmodified phage.
A plasmid pNP40 originating in Streotococcus lactis subsp. diacetvlactis DRC3 has been described by McKay L. L. and Baldwin K. A. (1). The mechanism associated with this plasmid was effective at 21.degree. and 32.degree. C. but not at 37.degree. C. Furthermore, evidence was presented that pNP40 was thermosensitive in its replication. Klaenhammer et. al. have described a plasmid pTR2030, originating in S. lactis ME2, which encodes a heat-sensitive phage-defence mechanism which limits the burst size and plaque size of specific phage without altering the efficiency of plaquing (e.o.p.) or the level of adsorption (2). This plasmid is described in European Specification No. 0208468A2. The pTR2030 mechanism was heat sensitive in an S. lactis background but not in S. cremoris hosts when challenged with isometric-headed phage (2, 3). The effect of high temperature on the activity of the pTR2030 associated mechanism against prolate-headed phage was not assessed. pTR2030 could be conjugally transferred to a range of S. cremoris strains (3, 4) and it was established that the plasmid conferred resistance to small isometric-headed phage. However, prolate- and large isometric-headed phage were either not inhibited in the transconjugants or exhibited a reduction in plaque size without a reduction in e.o.p. (5). The presence of pTR2030 in a lysogen was shown to be unable to inhibit prophage induction in S. cremoris R1 but it did prevent the lysogenic phage from infecting a pTR2030-containing prophage cured derivative S. cremoris R1 (6). It was suggested that the phage resistance mechanism acts at the cell surface or membrane to prevent small isometric-headed phage DNA passage into the host cell or inhibits early events required for lytic replication of externally infecting phage (6). Sanders et al. have devised a conjugal strategy for the construction of fast acid-producing, bacteriophage-resistant lactic streptococci for use in dairy fermentations by introducing pTR2030 into lactose-utilising, antibiotic-sensitive recipient strains (7). This report also describes the use of deletion analysis to locate the region of pTR2030 encoding phage resistance and the cloning of a specific HindIII fragment in E. coli using the pBR322 vector plasmid. A 150 bp region of pTR2030 was sequenced and 19 base pairs from within this region were chemically synthesized to be used as a very specific probe to identify strains harbouring pTR2030.
Reference 4, 5, 6 and 7 and European Specification No. 0208468A2 were published after the first priority date of the present application.
Although there has been considerable progress in the study of plasmid-encoded phage insensitivity mechanisms, there is still a definite need for more potent systems. In particular, a heat insensitive mechanism active against virulent prolate-headed phage in S. cremoris and S. lactis backgrounds is highly desirable for use in the food industry.
It is an object of the present invention to provide plasmids or DNA fragments which encode phage-insensitivity in species useful in cheese manufacture and other areas of the food industry.